Unintended spread of a biosafety level 2 recombinant retrovirus

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RETROVIROLOGY

Short report

Unintended spread of a biosafety

level 2 recombinant retrovirus

Stang1, beth Petrasch-Parwez2, Sabine

Brandt1, Rolf Dermietzel2, Helmut E Meyer3, Kai Stühler3,

Sven-T Liffers3, Klaus Überla1 and Grunwald1

1 Department of Molecular and Medical Virology,

Ruhr-University Bochum, D-44780 Bochum, Germany

2 Department of Neuroanatomy and Molecular Brain Research,

Ruhr-University Bochum, D-44780 Bochum, Germany

3 Medical Proteome Center, Ruhr-University Bochum, D-44780

Bochum, Germany

Retrovirology 2009, 6:86

doi:10.1186/1742-4690-6-86

The electronic version of this article is the complete one and

can be found online at:

http://www.retrovirology.com/content/6/1/86

Received: 23 April 2009

Accepted: 22 September 2009

Published: 22 September 2009

© 2009 Stang et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of

the Creative Commons Attribution License

(http://creativecommons.org/licenses/by/2.0), which permits

unrestricted use, distribution, and reproduction in any medium,

provided the original work is properly cited.

Abstract

Background

Contamination of vertebrate cell lines with animal retroviruses

has been documented repeatedly before. Although such viral

contaminants can be easily identified with high sensitivity by

PCR, it is impossible to screen for all potential contaminants.

Therefore, we explored two novel methods to identify viral

contaminations in cell lines without prior knowledge of the

kind of contaminant.

Results

The first hint for the presence of contaminating retroviruses in

one of our cell lines was obtained by electron microscopy of

exosome-like vesicles released from the supernatants of

transfected 293T cells.

Random amplification of particle associated RNAs (PAN-PCR)

from supernatant of contaminated 293T cells and sequencing

of the amplicons revealed several nucleotide sequences

showing highest similarity to either murine leukemia virus

(MuLV) or squirrel monkey retrovirus (SMRV).

Subsequent mass spectrometry analysis confirmed our

findings, since we could identify several peptide sequences

originating from monkey and murine retroviral proteins.

Quantitative PCRs were established for both viruses to test

currently cultured cell lines as well as liquid nitrogen frozen

cell stocks.

Gene fragments for both viruses could be detected in a broad

range of permissive cell lines from multiple species.

Furthermore, experimental infections of cells negative for

these viruses showed that both viruses replicate rapidly to

high loads.

We decided to further analyze the genomic sequence of the

MuLV-like contaminant virus.

Surprisingly it was neither identical to MuLV nor to the novel

xenotropic MuLV related retrovirus (XMRV) but showed 99%

identity to a synthetic retrovirus which was engineered in the

1980s.

Conclusion

The high degree of nucleotide identity suggests unintended

spread of a biosafety level 2 recombinant virus, which could

also affect the risk assessment of gene-modified organisms

released from contaminated cell cultures.

The study further indicates that both mass spectrometry and

PAN-PCR are powerful methods to identify viral contaminations

in cell lines without prior knowledge of the kind of

contaminant.

Both methods might be useful tools for testing cell lines

before using them for critical purposes.

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The full text can be found at:

http://www.retrovirology.com/content/6/1/86

A PDF version can be found at:

http://www.retrovirology.com/content/pdf/1742-4690-6-86.pdf